Sample concentrator

ABSTRACT

To concentrate a material by separating it from a diluting medium, the combination of the material and medium is placed in one of two wells in the bottom of a plastic sample concentrator cell, with each well being closed at its bottom end by a different cellophane membrane in contact with a buffer solution in a different one of two buffer compartments. A buffer solution also connects the combination of material and medium in one well and the cellophane bottom of the other well within the sample concentration cell through a recess in the bottom of the sample concentration cell. A potential is applied across the two buffer compartments to cause the material to migrate by electrophoresis from the medium in one well, through the buffer in the sample concentrating cell and into the other well, where it is concentrated against the cellophane membrane for easy removal by pipetting.

This is a division of application Ser. No. 781,176 filed Nov. 25, 1977.

This invention relates to methods and apparatuses for concentratingsamples.

Materials which have been separated one from the other inchromatographic or other processes are often combined with a medium usedin the separation. For example, proteins separated by certaindensity-gradient centrifugation processes are in a sucrose solutionafter being collected and proteins separated by certain electrophoresisprocesses are in a polyacrylamide gel after being separated. Thematerials are separated from the diluting medium and concentrated beforebeing analyzed or used. Moreover, it is, under some circumstances,desirable to concentrate samples before separating the molecular speciesof the samples by chromatography.

In the prior art, these materials are generally separated andconcentrated by dialysis and evaporation if they are in a liquid mediumand by elution followed by dialysis and evaporation if they are in a gelmedium. These prior art methods and apparatuses for separating thedesired materials from a combination of the materials and a medium havethe disadvantage of being relatively slow, requiring twelve or morehours under some circumstances.

Attempts have been made in the prior art to reduce the time required forconcentrating some dilute proteins by applying an electric field acrossthe dilute protein to electrophores the protein from a first side of afilter paper separator, which serves as an anticonvection element to asecond side where it is collected in concentrated form. A prior artmethod using this principle is disclosed in U.S. Pat. No. 3,079,318.This prior art method has the disadvantages of not providing sufficientseparation, being relatively slow and losing some of the proteins in thefilter paper.

Accordingly, it is an object of the invention to provide a novel methodfor concentrating samples by separating them from a diluting medium.

It is a further object of the invention to provide a novel apparatus forconcentrating samples.

It is a still further object of the invention to provide a relativelyrapid method for concentrating samples by electrophoresis.

It is a still further object of the invention to provide an apparatuscapable of concentrating samples in a relatively short time.

It is a still further object of the invention to separate andconcentrate a sample from a larger volume of unwanted material in acontinuous flow process.

In accordance with the above and further objects of the invention, aplastic sample concentration cell includes two wells in its bottomsurface, each of which is closed by a porous membrane of a material suchas cellophane (reconstituted cellulose). One of the wells is larger thanthe other and the two wells are connected by a recess in the insidebottom wall of the sample concentration cell.

In one embodiment, the sample concentration cells are adapted to bemounted to an electrophoretic cell having four side-by-side buffercompartments, with one of the wells contacting the buffer in a firstinner buffer compartment and the other well contacting the buffer in asecond outer buffer compartment of the electrophoretic cell, the firstand second buffer compartments being insulated from each other exceptthrough the sample concentration cells and having a first outer buffercompartment adjacent to one side of the first inner buffer compartmentand a second outer buffer compartment adjacent to one side of the secondinner buffer compartment. The path between the cells is advantageouslycooled such as by tubes carrying a fluidic coolant.

In one mode of operation, the sample is pipetted into the larger of thewells and the bottom of the recess in the sample concentrating cell iscovered with a buffer solution to permit an electrical current to flowfrom the buffer in one of the buffer compartments through one of theporous membranes, the well containing the sample, the buffer solution inthe recess in the sample concentrating cell, the porous membrane of theother well and into the buffer solution in the second buffercompartment. In another mode of operation, the entire sampleconcentrating cell is filled by the sample solution and in still anothermode of operation, the sample solution flows continuously through thecell and the concentrate is removed.

In each mode of operation, a potential is applied across the buffercompartments, causing the material in the well containing the sample tomigrate by electrophoresis within the sample concentrating cell to theother well, where it is forced against the cellophane membrane at thebottom of the well, while the diluting medium remains in the first well.Care is taken to avoid the excessive dilution of the sample by thebuffer except for the movement of the electrophoresed material.Generally, this dilution may be avoided by selecting a buffer having aspecific gravity that is not substantially greater than that of thesample.

The starting material may be any material that includes: (1) relativelylarge molecular species which migrates in the presence of an electricfield; (2) smaller ions which can pass through the porous membrane; and(3) materials which do not migrate with any substantial velocity in thepresence of the electric field. The wells serve to separate the startingmaterial from the concentrated material but other structures may beutilized for the same purpose, and, for some combinations, no particularstructure is necessary, such as the case where material is moved fromone gel into another gel at a different location. In one embodiment, thelarger well is elongated and the dilute sample is continuously insertedin one side in a heavy solution beneath the surface of the buffer andremoved from the other side during operation to provide a continuousflow of dilute sample for concentration in the smaller well. Moreover,the concentrate may also be continuously removed.

The methods and apparatuses of this invention have the advantages ofoperating relatively rapidly and being capable of moving a material fromone medium into a protective medium when this is desired. Moreover, inone embodiment, a continuous flow of a dilute sample may beconcentrated.

The above noted and other features of the invention will be betterunderstood from the following detailed description when considered withreference to the accompanying drawings in which:

FIG. 1 is a simplified perspective view of a sample concentrator inaccordance with an embodiment of the invention;

FIG. 2 is a plan view of the embodiment of FIG. 1;

FIG. 3 is a sectional view through lines 3--3 of FIG. 2;

FIG. 4 is an elevational view of a sample concentrating cell used in thesample concentrator of FIG. 1;

FIG. 5 is a bottom view of the sample concentrating cell shown in FIG.4;

FIG. 6 is a plan view of the sample concentrating cell shown in FIG. 4;

FIG. 7 is a plan view of another sample concentrating cell usable in thesample concentrator of FIG. 1;

FIG. 8 is a side elevational view of the sample concentrating cell ofFIG. 7;

FIG. 9 is a back elevational view of the sample concentrating cell ofFIG. 7;

FIG. 10 is a front elevational view of the sample concentrating cell ofFIG. 7;

FIG. 11 is a perspective view of a cylindrical volume-reducing insertuseful in the embodiment of FIGS. 4, 5 and 6;

FIG. 12 is a perspective view of a parallelepiped-shaped volume-reducinginsert useful in the embodiment of FIGS. 7, 8, 9 and 10;

FIG. 13 is a perspective view of another embodiment of sampleconcentrating cell; and

FIG. 14 is an exploded perspective view of another embodiment of aportion of a sample concentrator.

In FIGS. 1 and 2 there is shown an early embodiment of a sampleconcentrator 10 having a sample concentrating cell 12 and anelectrophoretic section 14, with the electrophoretic section 14including an electrophoretic cell 16 and an electrical power supply 18.The sample concentrating cell 12 rests within the electrophoretic cell16, having its bottom side in contact therewith and the electrical powersupply 18 is electrically connected to the sample concentrating cell 12through the electrophoretic cell 16.

To provide an electrical potential to the electrophoretic cell 16, theelectrical power supply 18 of the electrophoretic section 14 includesfirst and second electrodes 20 and 22, which are insertable into theelectrophoretic cell 16, with the electrode 20 being electricallyconnected to the positive output terminal of the DC power supply 18 andthe electrode 22 being electrically connected to the negative outputterminal of the DC power supply 18, by electrical cords. The powersupply 18 and the electrodes 20 and 22 are of any suitable type used forelectrophoresis, many brands of which are sold and which commonlyprovide potentials of up to approximately 2000 volts DC.

To provide the electrical potential to the sample concentrating cells 12in the embodiment of FIG. 1, the electrophoretic cell 16 includesplastic wall portions forming two buffer compartments 26 and 28 cooledby water coils 33 with the two compartments 26 and 28 being separatedfrom each other by a vertical, elongated, separating wall 30 whichextends the length of the buffer compartments 26 and 28 within fourenclosing side walls 32 and a bottom 34 of the electrophoretic cell 16.The walls 30 and 32 and the bottom 34 of the electrophoretic cell 16 areof any suitable plastic such as polycarbonate suitable for containing abuffer solution.

The separating wall 30 may be cored for cooling and is dimensioned witha width that tightly receives the sample concentration cell 12.

In another embodiment described hereinafter, two outer compartments areformed between the walls 32 and the separating wall 30 in thecompartments 26 and 28 by two semipermiable membranes which extend thelength of the buffer compartments parallel to the separating wall 30,with one membrane being located between the separating wall 30 and oneside wall portion and the other membrane being located between theseparating wall 30 and an opposite side wall portion. The outer buffercompartments contain buffer solution having a higher concentration thanthe buffer solution in the inner compartments. Each of the electrodes 20and 22 make electrical contact with the buffer solution within adifferent one of the outer buffer compartments.

As best shown in FIGS. 2 and 3, the separating wall 30 includes withinit a chamber having tubing 31 which extends through the walls 32 whereit may be connected to a source of coolant to cool the upper end of theseparating wall 30 and the concentrating cell 12 that rests upon it.

While specific separate electrophoretic and sample concentrating cells12 are shown in FIGS. 1 and 2, other configurations of sampleconcentration are possible such as configurations havingintegrally-formed sample concentrating and electrophoretic cells.Generally, the electrophoretic cell should have electrodes or structureto receive electrodes which apply a potential difference across portionsof the sample concentrating cells, and advantageously have compartmentsfor a buffer solution which is useful in providing a flow of ions in thefield caused by the electric potential.

In FIG. 4, there is shown one embodiment of sample concentrating cell 12having an outer vertical cylindrical wall 36 and a bottom wall 38forming a compartment 37 (FIGS. 4 and 6) suitable for holding fluids.

As shown in FIG. 4, the bottom wall 38 includes a groove 40 having awidth substantially the same size as the width of the separating wall 30so that the sample concentrating cells 12 rest upon the separating wall30 (FIG. 2) with the top edge of the wall 30 witting within the groove40 to hold each sample cell in place and properly aligned.

Two side legs 41A and 41B project from the walls of the groove 40 neartheir bottom and spaced from the top wall of the groove to preventbuffer solution from moving up the separating wall 30 to electricallyshort the two buffer compartments 26 and 28. The projections space thewalls of the groove 40 from the sides of the separating wall 30 to avoidcapillary action. Of course, projections on the wall 30 or channels orother devices may be used for this purpose as well.

Although only one cell is shown resting on the separating wall 30 inFIGS. 1 and 2, it is obvious that different numbers of such cells mayrest on the wall with portions of the cells extending to either side ofthe wall where they are in intimate contact with the buffer solution inthe buffer compartments 26 and 28, the separating wall 30 advantageouslybeing lower than the outer wall 32 for this purpose. When more than onecell is used, care must be taken to control the parallel electricalpaths formed thereby to achieve the desired results.

To receive a potential applied to a buffer solution within the buffercompartments 26 and 28, the bottom wall 38 (FIGS. 4 and 6) of the sampleconcentrating cell 12 includes two apertures 42 and 44 connected by arecess 45, each aperture being positioned on a different side of thegroove 40. As best shown in FIG. 4, the aperture 44 extends downwardlythrough a cylindrical projection from the bottom wall 38 to provide alonger aperture. The apertures are circular in cross-section and closedat the bottom surface by a porous membrane such as a cellophane membraneto form in the inner bottom wall of the separating cells 12 (FIG. 6)cylindrical walls extending downwardly into the bottom wall 38 and beingclosed at their bottom surfaces by membranes where engagement is madewith buffer in the buffer compartments 26 and 28.

The well 42 has a larger cylindrical bottom area than the well 44 and isnormally positioned over one of the buffer compartments 26 and 28,receiving the electrode 20 that has a positive potential appliedthereto. The smaller well 44 is positioned over the other buffercompartment which receives the electrode 22 having a negative potentialapplied thereto. The large well 42 is the sample well or startingmixture well and the smaller well 44 is the receiving well or theconcentrate well. For some applications, the larger well 42 may bepositioned over the compartment receiving a negative potential and thewell 44 over the compartment receiving a positive potential.

While the sample concentrating cells 12 shown in FIGS. 4-6 are generallycup shaped, they need not be; but only need to include a path whichpermits ion flow between two other sections one of which contains aconcentrate of a first material having a relatively high migration ratein the presence of an electrical field and which cannot readily passthrough the semipermeable membrane closing the well 44 and the other ofwhich contains the starting mixture containing the first materialdiluted with a second material which second material either has a lowerrate of or no migration in the presence of the electric field so thatthe concentrate is movable from the starting or sample section to thereceiving or concentrate section or can pass through the semipermeablemembrane slowing the well 44. To separate some materials, for example,separate sample and concentrate wells are unnecessary and a flat bottomsurface or single trench is sufficient such as when separating theconcentrate from a gel since the gel remains separated from theconcentrate portion even though it rests above or at the same level ofthe sample separating cell as the concentrate.

In FIGS. 7-10, there is shown a sample concentration cell 46 having thegeneral shape of an open top right regular parallelepiped withupstanding vertical walls 48 and a bottom wall 50. As best shown inFIGS. 8 and 9, the bottom wall 50 on the bottom outside thereof includesthe rectangular recess 52 which is substantially of the same shape andsize as the top of the separating wall 30 (FIG. 2) so as to permit thesample concentrating cell 46 to be mounted thereto. Within therectangular recess 52 are inwardly extending legs 53 similar to theprojections 41A and 41B in FIG. 4 to space the walls of the recess 42sufficiently far from the sides of the separating wall 30 to avoidcapillary movement of buffer up the separating wall 30 from thecompartments 26 and 28 to electrically short these compartments.

In the bottom wall on one side of the rectangular groove 52 is a firstaperture or well 54, shaped as a right regular parallelepiped and on theopposite side is a smaller aperture or well 56, having the same generalshape with each of the apertures 54 and 56 being sealed to the bottomwall 50 by a different cellophane membrane or different portions of thesame membrane to form a porous contact between the apertures 54 and 56and a buffer at 50A and 50B respectively. When the sample concentratingcell 46 is mounted in intimate contact with the buffer in respectiveones of the buffer chambers 26A and 28A, the apertures 54 and 56 withtheir respective membrane bottoms form the sample well 54 andconcentrate well 56 respectively. The sample well 54 is connected to theconcentrate well by a connecting recess 58 in the top surface of thebottom wall 50 to permit the flow of the material being separatedbetween the sample cell and the concentrate cell and to receive acovering buffer solution to facilitate this flow.

To permit a large volume of dilute sample to be concentrated, the sampleconcentrator 46 includes an inlet 60A and an outlet 62A, each beingwithin the wall 48 on a different side of the sample well 54. The inlet60 includes a downwardly-extending cylindrical recess 61A communicatingwith a first end of the sample well 54 near its bottom through a tube63A and with an inlet tube 65A at a height above the top of theconnecting recess 58 and the outlet 62 includes a downwardly-extendingcylindrical recess 61B communicating with the second end of the samplewell 54 near its bottom through a tube 63B and with an outlet spout 65Bat a height above the top of the connecting recess 58.

These connections are arranged to permit the continuous flow of a dilutesample through the sample well 54 at a level below the buffer thereinfor continual removal of the material to be concentrated to theconcentrate well 56, with the spout 65B being sufficiently large toavoid siphoning action. Preferably the buffer has a lower density thanthe sample so the sample flows across the top of the membrane with thebuffer floating on top of the sample.

Similar connections including an inlet 60B, an outlet 62B, recesses 61Cand 61D, tubes 63C and 63D and inlet tubes 65C and 65D provide forcontinuous flow of concentrate through the concentrate well. This ispossible because the concentrate is more dense than the buffer and flowsalong the bottom of the concentrate well.

Generally, the semiporous membranes that close the sample andconcentrate wells extend along a portion of the bottom surfaces of theconcentration cells on each side of the groove 40 or 52 to cover thebottoms of the wells 42, 44, 56 and 58 for convenient sealing andfabrication of the sample and concentration wells. In one embodiment,the semiporous membranes are held in place by acrylic rings that extendpart way into the grooves 40 and 52 and surround the walls of the sampleconcentrate cells, holding the edge of the membrane between them and thewalls. In this embodiment the rings also space the walls of the cellfrom the separating wall 30 to prevent capillary action. The remainderof the sample concentration cells 12 and 46 are of any suitable plasticsuch as polycarbonate or acrylic.

In the embodiment of FIGS. 7-10, structure is shown to permit continuousin line operation to concentrate large amounts of dilute sample. Ofcourse, this type of structure can also be used in other embodiments ofsample concentration cells such as the embodiment of FIGS. 4-6.

In FIGS. 11 and 12, there are shown two differentconcentrate-well-volume reducers 68 and 70, each having a different oneof the elongated handles 72 and 74 respectively and a different one ofthe well inserts 76 and 78 respectively. These volume reducers are indifferent sizes to fit with concentrate wells, with the reducer 68fitting within a cylindrical well and the reducer 70 within wells have arectangular cross-section to reduce the amount of buffer above theconcentrate in the well for ease in removing the concentrate.

Before operating the sample concentrator 10, a suitable number of sampleconcentrating cells 12 or 46 are positioned on the separating wall 30with the sample well 42 or 54 being positioned over the buffercompartment 26 and the concentrate wells 44 or 56 being positioned overthe buffer compartment 28. The separating wall 30 is received by thegrooves 40 or 52 of the sample concentrating cells 12 or 46. If theconcentrate is expected to be substantially lower in volume than thevolume of the concentrate well, a reducer 68 or 70 is inserted into theconcentrate well to reduce the volume of the buffer in the well.

With the cells mounted to the separating wall 30, buffer is applied tothe buffer compartments 26 and 28 and the electrodes 20 and 22 areinserted into the buffer, with the buffer extending up to the top of theseparating wall 30 but not over the wall so that the compartments 26 and28 are electrically insulated from each other except through the samplecells 12 or 46 which extend into the buffer solutions. The electrode 20is electrically normally connected to the positive output terminal andthe electrode 22 is normally electrically connected to the negativeterminal of the power supply 18. A coolant is circulated through thetube 31 of the eparating wall 30 so that a relatively large current maybe used for rapid separation of the concentrate from the sample withoutoverheating the concentrate. The temperature of the coolant is selectedin accordance with the need to maintain a low temperature.

In the embodiment of FIGS. 4-6, the sample is inserted into the samplewells 42 of the sample concentrating cells 12 and a buffer solution isapplied over the sample wells 42, concentrate wells 44 and recesses 45to connect the sample wells 42 to the concentrate wells 44 through therecesses 45. In the embodiment of FIGS. 7-10, connections are made tothe inlet tube 65A and outlet tube 65B to apply a continuous flow ofsample therethrough, with a buffer solution being placed over the samplecompartments 54, the recesses 58 and concentrate compartments 56 to forman electrical connection therebetween.

With this arrangement, a positive potential normally is applied to theelectrode 20 and normally a negative potential to the electrode 22 toprovide a path for electrical current from the electrode 20 to theelectrode 22. Generally the sample concentrating cells are arranged sothat the current flows through the buffer solution in the buffercompartment 26, through the cellophane bottom of the concentrate well,through the buffer solution within the sample cups, downward through thecellophane bottom of the sample well, into the buffer solution withinthe compartment 28 and finally to the electrode 22. This causes themigration of negative ions from the sample well to the concentrate welland positive ions to the sample well with small ions passing through thecellophane bottom of the concentrate well and into the buffer solutionin the buffer compartments while the concentrate to be separated is heldby the cellophane membrane to be gathered in the concentrate well andthe substance which does not migrate remaining in the sample well. Thisarrangement of concentrating cells is used with negatively chargedproteins and a reverse arrangement would be used to concentratepositively charged substances.

The connecting passageways (45 in FIGS. 4, 5 and 6 and 58 in FIGS. 7-10)restrict the flow of concentrate to an area having a relatively uniformfield and ion flow to avoid its being deposited at a location betweenthe sample well and concentrate well. To aid in confining the ion flowto the area of strong uniform field strength, the recesses 45 and 58conform to the sizes of the wells adjacent to their ends and slopeuniformly between their ends in the manner of the electric field.

Generally, the concentrate is a protein which is to be separated and isdiluted by another material such as a sucrose solution of the type usedin density-gradient centrifugation. Of course, other materials such asglycerol may form the base instead of sucrose as well as many other ofthe usual materials used in chromatography.

Although the preferred embodiment contemplates the removal of proteinsfrom sucrose or the like material and its deposition into a well whereit is to be held at the bottom of the well by gravity, other materialsmay be separated from a gel such as polyacrylamide gel and deposited inanother location in the sample concentrating cell or may be moved intoanother material. Similarly, the concentrate may be moved from onematerial such as a sucrose of one density and into another material suchas a more dense sucrose, glycerol or some other material useful infurther preparation of the sample or in analysis of the sample.

When the sample concentrating cell is being used to transfer a samplematerial from one substance to another, the sample material may or maynot be more concentrated in the new substance. For example, the transfermay be the transfer of a protein from sucrose to glycerol and theconcentration of the protein in the glycerol may be lower, the same, orhigher than it was in the sucrose. Moreover, the sample concentrator maybe used to transfer some species of materials through the walls of theseparating cell and retain others in the cell.

While the sample concentrating cells 12 or 46 are intended primarily toseparate the material that is being studied from the medium that it isin, which medium is present because it was used in the process ofseparating one molecular species from another or in receiving aseparated molecular species from another material for collecting in acommercial sample collector, it may be desirable to move it into anothermaterial because it has been found that some proteins have activeradicals attached to them which should not be exposed into theatmosphere or which should be protected in one manner or another. Bymoving the concentrate into a protective substance, further informationmay be obtained about the nature of the concentrate.

Once the concentrate has been separated from the medium of the startingmaterial, it may be removed by a pipette for further study in anycommercial analyzing apparatus or for use as a preparative material. Toremove the material, the buffer is pipetted from the recesses of thesample concentrator cell. The volume reducer is removed, leaving only asmall amount of buffer in the concentrate well some of which may beremoved, after which the concentrate is removed by pipetting.

In FIG. 13, there is shown another embodiment 80 of a sampleconcentrating cell having an outer vertical cylindrical wall 82 and abottom wall 84 forming a compartment 86 in a manner similar to theembodiment of FIGS. 4-6. This embodiment includes an upwardly openinggroove formed by a trough-shaped recess 88. The sample concentratingcell 80 rests upon the separating wall (31 in FIG. 2, 108 in FIG. 14)with the top edge of the wall 31 being below the recess 88 and thus doesnot have a recess into which the wall portion fits as do the embodimentsof FIGS. 4-10. The recess is omitted to reduce problems with capillaryaction from the buffer compartments.

To receive the potential difference applied across the buffer solutions,two cylindrical wells 90 and 92 extend downwardly from the trough 88,with the well 90 being slightly longer and with the well 92 including asmaller cylindrical projection 94 extending downwardly to substantiallythe same length as the well 90, with the well 92 being closed except forcommunication with the cylindrical projection 94. In the embodiment ofFIG. 13, the well 90 serves as the sample well or starting mixture welland the well 92 serves as the receiving well or concentrate well, withthe concentrate being moved generally downwardly into the projection 94which is smaller in diameter so as to have a more concentrated field.The bottoms of the wells are, of course, closed by a porous membrane inthe same manner as the embodiment of FIGS. 4-6.

To aid in supporting the sample concentrating cell 80 about a separatingwall, four legs 96A-96D extend downwardly from the bottom 84 of thesample concentrating cell, with adjacent legs 96A and 96B being joinedby a plastic strip closer to the bottom wall 84 than the bottom of therecess 88 and with the legs 96C and 96D being joined by a correspondingstrip.

The legs 96A-96D are thin plastic downwardly extending members which aresections of a cylinder and which have edges aligned with the inner edgesof the wells 90 and 92 with the inner edges of the legs 96A and 96Dbeing aligned with the inner edge of the tube 90 and the inner edges ofthe legs 96B and 96C being aligned with the inner edge of the well 92.With this arrangement, the opposite sides of the separating wall contactthe inner edges of the legs 96A and 96D on one side and 96B and 96C onthe other side. The legs are spaced to support the cell 80 on theseparating wall (30 in FIG. 2 and 108 in FIG. 14) by passing along thesides of the wall.

In FIG. 14, there is shown an exploded perspective view of anotherembodiment of electrophoretic cell 98 having a buffer compartmentsection 100 and a cover and electrode section which fits over and closesthe buffer compartment section 100.

To provide for buffer compartments, the buffer compartment section 100is generally parallelepiped in shape having a bottom wall 104, four sidewalls 106A-106D and an open top, with the four side walls and the bottomwall forming a compartment for holding buffer solutions. To supportsample concentrating cells, a center separating wall 108, shaped as aparallelepiped extends upwardly from the bottom wall 104 to a heightless than the side walls 106A and 106C which it intersects to divide theopen portion of the buffer compartment 102 into two sections, thuspermitting the sample concentrating cells to rest on top of theseparating wall 108 in a manner analagous to the manner in which theseparating cells rest on the separating wall 31. The separating wall 108is hollow and may support a plurality of cooling coils.

To separate each of the two sections of the buffer compartment section100 on each side of the separating wall 108 into two buffercompartments, there is formed on the bottom wall 104 and side walls 106Aand 106C on a first side of the separating wall 108, mounting tracks110A and on the other side of the separating wall 108, mounting tracks110B, with the mounting tracks 110A and 110B each including a recess inthe bottom wall 104 and the side walls 106A and 106C bordered on eachside by upwardly extending ridges.

The recesses and ridges forming the mounting tracks 110A are shaped toreceive a first separating membrane 112A and the recesses and ridgesforming the separating tracks 110B are shaped to receive a secondseparating membrane 112B and for this purpose extend parallel to theseparating wall 108. The mounting tracks and separating membranesseparate two outer high concentrate compartments from two inner lowconcentrate compartments.

To mount electrodes within the buffer solutions in the outer highconcentrate compartments, the compartment formed between the separatingmembrane 112A and the side wall 106D between walls 106A and 106Cincludes in the bottom wall 104 two cylindrical elevated bosses 114A and114B and a large upwardly extending boss 114C which extends above theheighth of the separating wall 108. Similarly, in the compartment formedby the separating membrane 112B and the side wall 106B between portionsof the walls 106A and 106C, the bottom wall 104 includes two upwardlyextending bosses 116A and 116B and a larger upstranding boss 116C whichextends above the heighth of the separating wall 108.

To provide electrical contact with the buffer solution in the outercompartment, a first electrode 118A is mounted to the bosses 114A-114Cand a second electrode 118B is mounted to the bosses 116A-116C. Thefirst electrode 118A and the second electrode 118B are each formed asthin metal strips shaped as L's, each with a long bottom section and anupstanding arm with an outwardly extending ear on it. The long bottomsection of the electrode 118A includes first and second apertures 120Aand 120B which are aligned with central apertures in the bosses 114A and114B and the outwardly extending ear has a third aperture 120C which isaligned with the aperture in the larger boss 114C.

The electrode 118A is designed to fit within the outer compartment withthe metal strip extending parallel to the separating wall 112A and theretaining apertures 120A-120C fitting over apertures in the bosses114A-114C. Similarly, the electrode 118B includes three apertures 122A,122B, and 122C each adapted to be aligned with a different one of thecentral apertures in the bosses 116A, 116B, and 116C for mounting in amanner similar to electrode 118A.

To hold the electrodes 118A and 118B in place: (1) first and secondretainers 124A and 124B each have a head portion larger than theapertures 120A and 120B and a shank which fits through the apertures120A and 120B and frictionally engages the apertures in the centers ofthe bosses 114A and 114B; and (2) retainers 126A and 126B have headslarger than the apertures 122A and 122B and shanks that fit throughapertures 122A and 122B and frictionally engage the center apertures inthe cylindrical bosses 116A and 116B. The electrodes 118A and 118B arepositioned against the bottom 104 of the base buffer compartment and theretainers 124A, 124B, 126A and 126B are positioned with their shankspassing through the apertures in the corresponding holes in the bossesto hold the electrodes 118A and 118B in place.

To provide electrical connection to the electrodes 118A and 118B fromoutside of the buffer section 100, first and second electrical contacts128A and 128B each include a corresponding one of the centralcylindrical sections 130A and 130B separating bottom cylindricalsections 132A and 132B and top pin shaped portions 134A and 134Brespectively. The bottom cylindrical sections 132A and 132B are of sucha size as to fit respectively through the apertures 120C and 122C andfit tightly within the central apertures in the bosses 114C and 116C.The central cylindrical sections 130A and 130B are larger than theapertures 120C and 122C so as to fit on top of the electrodes 118A and118B when mounted in position and the top pin shaped portions 134A and134B make electrical connection outside of the buffer solution toconductors as will be described hereinafter. The bottom cylindricalportions 132A and 132B have central openings in their bottom surfacewhich permit easy swedging of the metal around the bottom of theupwardly extending bosses 114A and 116A to hold the electrodes in place.

The separators 112A and 112B are plastic members of white polypropylenewhich are folded over and include windows of a semi-permeable materialthat allow electrical current to pass between the compartments butmaintain the high concentration compartment separate from the lowerconcentration compartment. The folder over sections include hook-likemembers in their ends which fit against the upstanding members in thetracks 110A and 110B respectively to hold the separators in place.

To cover the sample concentrator when it is in use, a plastic cover 136is shaped as a parallelepiped with downwardly extending walls 138 whichfit around the walls of the base buffer section 100 and a plastic topportion 140. To provide an outlet for fluids which may be useful forsome applications, a hooded opening 142 is provided which fits within anotch in the wall 106 of the buffer section 100. To permit the injectionof samples into sample cells and the removal of concentrate, there are aplurality of openings 144 in the top of the cover.

To permit electrical connection through the cover 136, the cover 136includes cylindrical members 146 and 148, each of which are hollowcylinders passing through the top of the cover 136 with a bottom partlyclosed to form a small central aperture and an open top. The smallcylindrical apertures in the bottoms of the cylinders 146 and 148 arealigned with the top pin-shaped connectors 134A and 134B respectivelywhen the cover 136 is properly positioned on the base buffer section 100so that the pins 134A and 134B extend upwardly therein to makeelectrical connection outside of the cover.

To connect the pins 134A and 134B to a power supply, first and secondfemale connectors 150A and 150B each have a different hollow conductiveconnecting member 152A and 152B with a recess adapted to fit tightlyabout a corresponding one of the pins 134A and 134B. The conductors 152Aand 152B are connected to cables 154A and 154B respectively and aremounted within an insulating member 156A and 156B. The insulating member156A fits comformably within the cylinder 146 and the insulating member156B fits comformably within the cylinder 148 so that the conductors150A and 150B may be inserted into the cylinders 146 and 148 to makeelectrical connection to the electrodes 118A and 118B in the powersupply.

As can be understood from the above description, the methods andapparatuses of this invention have the advantages of being able toquickly concentrate dilute material with high recovery.

While a preferred embodiment has been described in some detail, manymodifications and variations in the preferred embodiment are possible inthe light of the above teachings. Therefore, it is to be understoodthat, within the scope of the appended claims, the invention may bepracticed other than as specifically described.

What is claimed is:
 1. Apparatus for separating at least one molecularspecies from a sample, comprising:a sample concentrating cell; saidsample concentrating cell having first and second sections; said firstsection including a member adapted to receive a sample; said secondsection including a membrane having pores sufficiently small to permitthe membrane to hold said one molecular species and being adapted toreceive at least one of the separated molecular species; wall means forat least partly confining said first and second sections and adapted tosupport a buffer solution; means for establishing an electricalpotential between the side of the sample and the side of said membranefarthest from each other.
 2. Apparatus according to claim 1 in whichsaid member includes a second membrane.
 3. Apparatus according to claim2 in which said first and second membranes are cellophane membranes. 4.Apparatus according to claim 3 in which:said sample concentrating cellincludes top, bottom and side surfaces; said first section includesinternal walls defining a first opening; said first-mentioned membranecloses said first opening at said bottom surface; said second sectionincludes internal walls defining a second opening; said second membranecloses said second opening at said bottom, whereby said sample is placedin one opening, a buffer solution communicates between the first andsecond openings and the separated molecular species is received in saidsecond opening.
 5. Apparatus according to claim 4 further comprising:anelectrophoretic cell; said electrophoretic cell including walls definingfirst and second buffer compartments; one of said walls defining saidfirst and second buffer compartments comprising a separating wallbetween said first and second buffer compartments; said first and secondcompartments including means for receiving different electrodes, wherebya potential difference may be established between said first and secondbuffer compartments; and said sample concentrating cell including meansfor mounting said sample concentrating cell to said separating wall withone membrane within said first compartment and the other membrane withinsaid second compartment.
 6. Apparatus according to claim 5 in which saidmounting means comprises internal walls defining a groove in the outsidesurface of said bottom of said sample concentrating cell adapted toreceive said separating wall.
 7. Apparatus according to claim 6 furtherincluding a recess in the inner bottom of said sample concentrating cellconnecting said first and second sections.
 8. Apparatus according toclaim 7 in which said internal walls defining a groove includeprojections whereby said internal walls are spaced from said separatingwall to avoid capillary motion of buffer solution to the top of theseparating wall.
 9. Apparatus according to claim 7 further includingremovable insert means for reducing the volume of said second opening.10. Apparatus according to claim 7 further including means forpermitting a continuous flow of said sample through said first location,whereby said first molecular species may be removed from a continuouslyflowing stream.
 11. Apparatus according to claim 2 in which:said sampleconcentrating cell includes top, bottom and side surfaces; said firstsection includes internal walls defining a first opening; said firstmentioned membrane closes said first opening at said bottom surface;said second section includes internal walls defining a second opening;said second membrane closes said second opening at said bottom surface,whereby said sample is placed in said first opening, a buffer solutioncommunicates between the first and second openings and the separatedmolecular species is received in the second opening.
 12. Apparatusaccording to claim 11 further comprising:an electrophoretic cell; saidelectrophoretic cell including walls defining first and second buffercompartments; one of said walls defining said first and second buffercompartments comprising a separating wall between said first and secondbuffer compartments; said first and second compartments including meansfor receiving different electrodes, whereby a potential difference maybe established between said first and second buffer compartments; andsaid sample concentrating cell including means for mounting said sampleconcentrating cell to said separating wall with one membrane within saidfirst compartment and the other membrane within said second compartment.13. Apparatus according to claim 12 in which said mounting meanscomprises a groove in the outside surface of said bottom wall of saidsample concentrating cell.
 14. Apparatus according to claim 13 furtherincluding a recess in the inner bottom of said sample concentrating cellconnecting said first and second sections.
 15. Apparatus according toclaim 14 further including means for enabling a continuous flow of saidsample through said first location, whereby said first molecular speciesmay be removed from a continuously flowing stream.
 16. Apparatusaccording to claim 2 further comprising:an electrophoretic cell; saidelectrophoretic cell including walls defining first and second buffercompartments; one of said walls defining said first and second buffercompartments comprising a separating wall between said first and secondbuffer compartments; said first and second compartments including meansfor receiving different electrodes, whereby a potential difference maybe established between said first and second buffer compartments; andsaid sample concentrating cell including means for mounting said sampleconcentrating cell to said separating wall with one membrane within saidfirst compartment and the other membrane within said second compartment.17. Apparatus according to claim 16 in which said mounting meanscomprises a groove in the outside surface of said bottom wall of saidsample concentrating cell.
 18. Apparatus according to claim 15 furtherincluding a recess in the inner bottom of said sample concentrating cellconnecting said first and second sections.
 19. Apparatus according toclaim 18 further including means for enabling a continuous flow of saidsample through said first location, whereby said first molecular speciesmay be removed from a continuously flowing stream.
 20. Apparatusaccording to claim 5 further including a recess in the inner bottom ofsaid sample concentrating cell connecting said first and secondsections.
 21. Apparatus according to claim 20 further including meansfor enabling a continuous flow of said sample through said firstlocation, whereby said first molecular species may be removed from acontinuously flowing stream.
 22. Apparatus according to claim 12 furtherincluding a recess in the inner bottom of said sample concentrating cellconnecting said first and second sections.
 23. Apparatus according toclaim 22 further including means for enabling a continuous flow of saidsample through said first location, whereby said first molecular speciesmay be removed from a continuously flowing stream.
 24. Apparatusaccording to claim 16 further including a recess in the inner bottom ofsaid sample concentrating cell connecting said member andfirst-mentioned membrane.
 25. Apparatus according to claim 24 furtherincluding means for enabling a continuous flow of said sample throughsaid first location, whereby said first molecular species may be removedfrom a continuously flowing stream.
 26. Apparatus according to claim 1further including means for enabling a continuous flow of said samplethrough said first location, whereby said first molecular species may beremoved from a continuously flowing stream.
 27. Apparatus according toclaim 26 in which said member includes a second membrane.
 28. Apparatusaccording to claim 27 in which:said sample concentrating cell includestop, bottom and side surfaces; said first section includes internalwalls defining a first opening; said first-mentioned membrane closessaid first opening at said bottom surface; said second section includesinternal walls defining a second opening; said second membrane closessaid second opening at said bottom surface, whereby said sample isplaced in said first opening, a buffer solution communicates between thefirst and second openings and the separated molecular species isreceived in the second opening.
 29. Apparatus according to claim 26further comprising:an electrophoretic cell; said electrophoretic cellincluding walls defining first and second buffer compartments; one ofsaid walls defining said first and second buffer compartments comprisinga separating wall between said first and second buffer compartments;said first and second compartments including means for mounting saidsample concentrating cell to said separating wall with one membranewithin said first compartment and the other membrane within said secondcompartment.